Mid−log phase cells (5 ml) were harvested, and cell pellets were stored at −80°C. For analysis of protein levels, pellets were thawed on ice and resuspended in 300 µl 2× SDS sample buffer along with 100 µl of glass beads. After being vortexed for 5 min at full speed, samples were heated to 100°C for 5 min, then loaded onto gels for Western blot analysis. Proteins were detected using (1:1000) anti-GFP antibody (Cell Signaling Technology); histone H4 was the reference (1:10000) (Abcam).
Histone h4
Manufactured by Abcam
Sourced in United Kingdom
Histone H4 is a core histone protein that is a fundamental component of the nucleosome, the basic unit of chromatin in eukaryotic cells. Histone H4 plays a crucial role in the packaging and organization of DNA within the cell nucleus.
Automatically generated - may contain errors
Lab products found in correlation
Histone h3, by Abcam (8 mentions)
β actin, by Merck Group (4 mentions)
Flag tag (flag), by Merck Group (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
H4k16ac, by Merck Group (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Immobilon chemiluminescent reagent, by Merck Group (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Topo 1, by Santa Cruz Biotechnology (2 mentions)
Hsc70, by Abcam (2 mentions)
Hsp90, by Santa Cruz Biotechnology (2 mentions)
H3k9 14ac, by Diagenode (2 mentions)
H4k12ac, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti gfp antibody, by Cell Signaling Technology (1 mentions)
Plakoglobin, by Merck Group (1 mentions)
H3k9ac, by Merck Group (1 mentions)
H3k4me2, by Merck Group (1 mentions)
H4k20me3, by Abcam (1 mentions)
Vimentin, by Merck Group (1 mentions)
23 protocols using histone h4
1
Protein Extraction and Western Blot
2
RNA Pulldown and Histone Analysis
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For RNA pull‐down, the procedure was performed with minor modifications.20 Briefly, biotin‐labeled RP11‐367G18.1 variant 2 was synthesis by in vitro transcription and then purified. The biotinylated RP11‐367G18.1 variant 2 was incubated with cell lysates at 4°C for 6 h. The streptavidin agarose beads were added into the mixture and incubated for 1 h. The beads were washed and boiled for western blot analysis. As previously described,18 proteins and histones extracted from the cells were analyzed by SDS–PAGE using antibodies against HIF‐1α, N‐cadherin (BD Biosciences), H4K5Ac, H4K8Ac, H4K12Ac, H4K20me3, H3K56Ac, histone H4, p300, Tip60, HBO1, HAT1, CBP (Abcam), E‐cadherin, histone H3 (Cell Signaling Technology), plakoglobin, H3K4me2, H3K9Ac, H4K16Ac (Millipore), vimentin (Sigma‐Aldrich), and β‐actin (Genetex).
3
Immunoblot Analysis of Histone Modifiers
4
Protein Expression Analysis by Western Blot
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Protein extraction and western blotting were performed as described previously28 (link). Briefly, cells were lysed on ice for 30 min in radio-immunoprecipitation assay buffer. The lysates were centrifuged at 12,000 rpm at 4°C for 15 min, the supernatants were collected, and protein concentrations were determined using bicinchoninic acid assay TIANGEN, Beijing, China). Equal amounts of protein were separated by 10% SDS-PAGE followed by electrotransfer onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, MA, USA). Membranes were blocked with 5% nonfat milk for 2 h and then incubated overnight at 4°C with diluted (1:1000) primary antibodies against MMP2, MMP9, ZEB1, Snail, Slug, Twist and β-actin were obtained from Cell Signaling Technology (MA, USA), and ACOT12, N-Cadherin, E-Cadherin, histone H3, histone H3ac, histone H4 and histone H4ac obtained from Abcam followed by incubation with the secondary antibody (1:5000, Cell Signaling Technology). An electrochemiluminescence detection system (GE, USA) was used for signal detection.
5
Immunofluorescence of Histone H4
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iMEFS were seeded on six-well plates containing coverslips. Cells were fixed with 4% paraformaldehyde, blocked with 5% BSA and primary antibodies against histone H4 (Abcam) were incubated overnight, washed and replaced with goat-anti-rabbit secondary antibody. Cells were mounted in Vectashield mounting media containing DAPI and imaged using a wide field fluorescence microscope.
6
Protein Expression Profiling by Western Blot
7
Quantification of Histone Acetylation
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Proteins were extracted from one million cells after 2.5 h of treatment with DMSO or HDACi using RIPA buffer supplemented with 1 M NaF, 10 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail. For detection of specific acetylation of histone H3 and H4, the following antibodies were used: Acetyl-Histone H3 Antibody Sampler kit #9927 (Cell Signaling), anti-H3K23 (D6Y7M, Cell signaling), -H4K5 (EP1000Y, Abcam), -H4K8 (EP1002Y, Abcam), -H4K12 (Abcam), -H4K16 (EPR1004, Abcam), and -Histone H4 (Abcam).
8
Visualizing BAF170 Levels in Differentiated ES Cells
9
Visualizing BAF170 Levels in Differentiated ES Cells
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ES cell-derived neuronal BAF170 protein levels were visualized by western blot. Crude nuclear extracts were prepared from D12 mutant cell lines and CRISPR controls. Extracts were also prepared from D0, D4, D8 and D12 wild type differentiating cells. Extracts were run on SDS/PAGE gels (4-12%), transferred to PVDF membranes and probed with primary antibodies to BAF170 (Active Motif, 1:1000) and Histone H4 (1:6000, Abcam). Membranes were washed, probed with HRP tagged goat anti-rabbit secondary antibodies, and visualized with Immobilon chemiluminescent reagent (Millipore) using a Chemidoc Touch imaging system (Bio-Rad).
10
Acetylation Assay for His-HAT1
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The in vitro assay for acetyltransferase activity of His-HAT1 was performed with or without aptamers. His-HAT1 (100 ng or 2.5 pmol) was incubated either with the structured aptamers in the selection buffer or only with the selection buffer for 30 min at 37 °C. Next, 10 µL of Ni-NTA Superflow was added and incubated for 1 h at 4 °C. After three washes with the selection buffer, 27 µL of reaction buffer (100 mM Tris-HCl pH 7.6 and 250 ng of histone H4 (Abcam, Cambridge, UK)) was added and incubated for 1 min at 37 °C. Reaction began with the addition of 3 µL AcCoA 10 mM (Roche, Madrid, Spain) in 30 µL of final volume. Finally, 15 µL of denaturing buffer (Tris-HCl 180 mM pH 6.8, SDS 9%, β-mercaptoethanol 6%, glycerol 15%, and bromophenol blue 0.025%) was added to stop the enzymatic reaction after 2 h of incubation.
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